CN104114692A - Method of forming a multilayer aggregate of objects - Google Patents
Method of forming a multilayer aggregate of objects Download PDFInfo
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- CN104114692A CN104114692A CN201380008319.2A CN201380008319A CN104114692A CN 104114692 A CN104114692 A CN 104114692A CN 201380008319 A CN201380008319 A CN 201380008319A CN 104114692 A CN104114692 A CN 104114692A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
- C12N2521/10—Sound, e.g. ultrasounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4094—Concentrating samples by other techniques involving separation of suspended solids using ultrasound
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
- G01N2015/142—Acoustic or ultrasonic focussing
Abstract
A method of forming a multilayer aggregate (120) of objects (O) in a channel (2) comprising a liquid (L), said method comprising: a) providing objects (O) at first (50) and second (60) superposed regions of the channel (2), b) obtaining first (110) and second (111) aggregates of objects (O), optionally by applying transverse acoustic waves, preferably stationary waves, within each region to objects (O), and c) bringing said first (110) and second (111) aggregates into contact to form said multilayer aggregate (120) of objects (O) by submitting said first (110) and second (111) aggregates to: - gravity in absence of acoustic waves, or to - acoustic waves, optionally stationary waves, inducing displacement of said first (110) and second (111) aggregates toward each other.
Description
Technical field
The present invention relates to form the method for the multilayer aggregate of object in the passage that contains liquid.
Background technology
Organizational engineering field is by biomaterial is combined with viable cell, for regeneration therapy proposes infusive approach.For example, in the reconstruction that is combined in many various functional organizations (, bone, artery and bladder) of novel biomaterial (support) and viable cell, some clinical successes are produced.But, due to inflammatory reaction with cause the biodegradable pathology fibrosis of ensuing support state, there is being called as the novel Method of Tissue Engineering of " cell sheets engineering science ", be somebody's turn to do " cell sheets engineering science " by two-dimentional cell sheets layering being constructed to 3-D (three-dimensional) functional organization, and do not use any biodegradable extracellular matrix surrogate.
However,, in the time relating to functional application, the main drawback of cell sheets is the mechanical property of their difference.In the time leaving culture surface, cell sheets generally shrinks, thereby causes transplanting size reduction.In clinical setting, for specific application, controlling specific transplanting size and dimension will be desirable.In addition, the fragility of cell sheets makes difficult treatment.Even when avoid based on support technology time, in mechanically effective reconstruction of firm and manipulable 3-D tissue of the method addressing these problems, still there is major obstacles.
Because regenerative medicine therapy becomes more advanced, therefore there is ever-increasing demand for the strategy of the enlarged culturing of developing cells and tissue to meet the demand of expectation.As response, for using automatic cytological and tissue culture system to produce concern, the success of this cell and tissue culture system depends on monitoring and control strategy.There are the various culture techniques that form in vitro 2-D (two dimension) aggregate and 3-D aggregate in suspension, for example, in the lip-deep 2-D cell inoculation of plastic/glass flat and rigidity, bead (pellet) is cultivated, spherical (spheroid) cultivates and hanging drop cultivation, stent support, liquid covering, revolving bottle and rotary type rotation technique [4].But these methods can be limited to formation or the mechanical accessibility difficulty of incubation time length, big or small unequal aggregate.Therefore, they can be not suitable for the standardized, fast and the manufacture of large-scale 2-D aggregate/3-D aggregate of the needed form of high throughput analysis.
The sound power of utilizing the standing acoustic waves place by acting in thin chamber to produce
can realize the particle of handling micron and submicron-scale.V is the volume of particle, and <e> is average acoustic energy, and k=2 π/λ is wave number,
for depending on the acoustics contrast factor of acoustical behavior of particle and suspension.
This chamber can be called as " acoustic resonator ".This syntonizer can comprise transmitting wall and reflecting wall.In the time that the relation of the thickness w of chamber harmony wavelength X is as shown in equation below: standing wave, can appear in w=n λ/2 (wherein, n is the number that is formed on the node in the thickness of chamber).According to acoustics contrast factor
(this acoustics contrast factor
for being defined as product ρ
ic
ithe function of specific acoustic resistance, wherein, ρ
iand c
ifor density and the velocity of sound of fluid or suspended particle), be subject to the particle in this sound field of force of variable thickness, can be pushed to node or the antinode of standing wave.
The technology of the organizational engineering that development needs for cosmonaut, also will attract people's attention.Under below-G conditions in space, due to the migration and not by guiding trestle or matrix naturally (aggregate and other tissue must in support or the growth of matrix place), therefore cell cultures is a challenge in reactor of the cell in substratum.Under weightlessness, form the further cost time of several days of aggregate.
Need a kind of method that allows to form fast 2-D cell aggregation and 3-D cell aggregation, the method can be favourable for the ordinary method of aforementioned cell cultures.
Need to obtain a kind of method that allows to form the multilayered structure that comprises organism (for example, cell).
Also need to obtain a kind of method of formative tissue simulation structure.
Also need to obtain the improvement technology of organizational engineering.
The object of the invention is to meet some or all in above-mentioned needs.
Summary of the invention
According to first aspect, the invention provides a kind of method that forms the multilayer aggregate of object in the passage that contains liquid, described method comprises:
A) provide object in the first overlap-add region and second overlap-add region of passage,
B) the first aggregate and second aggregate of acquisition object, and
C) by making described the first aggregate and described the second aggregate:
Zero in the time not there is not sound wave, is subject to action of gravity, or
Zero is subject to sound wave, the standing wave effect alternatively of inducing described the first aggregate and described the second aggregate to move towards each other,
Described the first aggregate is contacted with described the second aggregate, and form the multilayer aggregate of described object.
The meaning of " aggregate of object " is the layer that meets the object of all following features:
-be included at least two object contacts in described layer, be especially included in the object contact of at least 10%, better 25%, preferably 50% in described layer, and
-in the time that at least one dimension in its transverse dimensions moves, described layer presents a string object at least a portion of its length.
The aggregate obtaining can be 2D aggregate or 3D aggregate.
The aggregate obtaining can planar extend, and comprises object side by side, is especially made up of object side by side.In this case, aggregate is called as 2D aggregate.
In another embodiment, the aggregate obtaining comprises a folded 2D aggregate.This embodiment is the example of 3D aggregate.
Aggregate is different from the only a line object along Axis Extension.In other words, a line object only comprises an object, only comprises an object and comprise a string object along its length on thickness on width.
The present invention can advantageously allow without damage to collect continuously aggregate.
The present invention can advantageously allow the formation of the multilayer aggregate of one species or different types of object.
The present invention can advantageously can control and sequencing form the cell aggregation (2D and/or 3D) with consistent size.
It is (homotype and/or special-shaped that the present invention can advantageously allow to form 2-D cell sheets simultaneously,, identical or different cell type), described 2-D cell sheets is by the mode with layering, the mode of invading with minimally positions, for example,, so that formative tissue simulation structure.
The present invention can advantageously allow to handle and control aggregate, and avoids the contact of the characteristic that changes them between object and the wall of passage.
The present invention also advantageously allows to obtain fast aggregate, especially the 3-D cell aggregation of object.
The present invention advantageously allows use device to obtain multilayer aggregate, and this device can relatively easily be manufactured, and can be used under aseptic condition.Can comprise standard ultrasound equipment with device in the method according to the invention.
A) can provide the layer at the first overlap-add region of passage and the object of the second overlap-add region in step, described layer is focused on and is obtained by water power alternatively.
A) being present in object in first area in step can have to be different from step and a) be present in size and/or the character of the object in second area.
In step b), by apply transverse sound wave in each region, standing wave alternatively, the first aggregate and second aggregate of acquisition object.
In modification, in step b), focus on by water power, can obtain the first aggregate and second aggregate of object.In this case, the object that forms aggregate can advantageously present surface interaction, and for example, electrostatic interaction, moves object more each other close.
According to an embodiment, stop sound field in step in c), the aggregate of object, because gravity contacts, therefore, is placed on above another one of described aggregate, for example, causes the tissue simulation structure of organized layering.
This embodiment can advantageously allow to form to suspend and be arranged to the cell of cell sheets, thereby avoids the contraction of cell sheets.This embodiment is also that minimum level ground is invasive, and can be convenient to process.
According to some embodiments, step c) comprises and makes aggregate be subject to acoustic wave action, and this sound wave has:
-different from the number of the sound wave joint of the sound wave of b) applying in step, save few sound wave than the sound wave of the sound wave of b) apply in step alternatively and save, and/or
-at least one sound wave joint, sound wave abdomen separately, the sound wave that the horizontal position of this at least one sound wave joint, sound wave abdomen is separately different from the sound wave of b) apply in step saves, the horizontal position of sound wave abdomen separately, and/or
-be less than the amplitude of the sound wave amplitude of b) applying in step.
According to a specific embodiment, there is at least first sound field generator and rising tone field generator along the length of passage, and:
-described first sound field generator and described rising tone field generator step b) and step c) launch sound wave, and
-described passage has the first wall and the second wall that lay respectively on described first sound field generator and described rising tone field generator opposite, described first wall and described the second wall have different thickness, make in this way the sound wave of c) applying in step have the node and/or the antinode that are positioned on described the second wall.
This embodiment can advantageously allow the multilayer aggregate of formed object to be positioned on one of them wall of passage, thereby is easy to separate.
According to a specific embodiment, there is at least first sound field generator and rising tone field generator along the length of passage, for being created on respectively step b) and step sound wave c), wherein:
-described first sound field generator and described rising tone field generator step b) and step c) launch the sound wave with identical substantially primary frequency, and
The width of-passage and/or thickness change, reduce alternatively at least a portion of its length, the sound wave that makes in this way c) to apply in step have the number of sound wave joint of the sound wave of b) applying from step different, save few sound wave than the sound wave of the sound wave of b) apply in step alternatively and save.
According to a specific embodiment, the primary frequency of the sound wave of c) applying in step can be different from the primary frequency of the sound wave of b) applying in step, and that the sound wave that makes in this way c) to apply in step has is different from the number of the sound wave joint of the sound wave of b) applying in step, save few sound wave than the sound wave of the sound wave of b) apply in step alternatively saves.
According to a specific embodiment, the present invention relates to a kind of method, the method comprises:
A) provide object in the first overlap-add region, the second overlap-add region and the 3rd overlap-add region of passage,
B) alternatively by apply transverse sound wave, especially standing wave to object in each region, obtain the first aggregate, the second aggregate and the 3rd aggregate of object, and
C) by making described the first aggregate, described the second aggregate and described the 3rd aggregate:
Zero in the time not there is not sound wave, is subject to action of gravity, or
Zero is subject to inducing the acoustic wave action of the power that described the first aggregate, described the second aggregate and described the 3rd aggregate move towards each other,
Described the first aggregate, described the second aggregate and described the 3rd aggregate are contacted, to form the multilayer aggregate of described object.
In specific implementations of the present invention, at least one in the first aggregate and the second aggregate and optional the 3rd aggregate comprises at least 100 objects, especially at least 250 objects, especially at least 500 objects, especially at least 750 objects, especially at least 1000 objects, especially at least 1500 objects.
In the preferred embodiment of the present invention, at least one in the first aggregate and the second aggregate and optional the 3rd aggregate comprises at least 100 cells, at least 250 cells especially, especially at least 500 cells, especially at least 750 cells, especially at least 1000 cells, especially at least 1500 cells.
In the preferred embodiment of the present invention, each in the first aggregate and the second aggregate and optional the 3rd aggregate comprises at least 100 objects, at least 250 objects especially, especially at least 500 objects, especially at least 750 objects, especially at least 1000 objects, especially at least 1500 objects.
In the preferred embodiment of the present invention, each in the first aggregate and the second aggregate and optional the 3rd aggregate comprises at least 100 cells, especially at least 250 cells, especially at least 500 cells, especially at least 750 cells, especially at least 1000 cells, especially at least 1500 cells.
As mentioned above, the present invention advantageously allows to obtain fast and easily cell aggregation, and the 3D structure of hundreds of cell preferably in seconds forms.
According to a specific embodiment, be less than or equal to 10m/s in intensity
2, be especially less than or equal to 9.5m/s
2, be especially less than or equal to 9m/s
2, be especially less than or equal to 8.5m/s
2, be especially less than or equal to 8m/s
2, be especially less than or equal to 7.5m/s
2, be especially less than or equal to 7m/s
2, be especially less than or equal to 6.5m/s
2, be especially less than or equal to 6m/s
2, be especially less than or equal to 5.5m/s
2, be especially less than or equal to 5m/s
2, be especially less than or equal to 4.5m/s
2, be especially less than or equal to 4m/s
2, be especially less than or equal to 3.5m/s
2, be especially less than or equal to 3m/s
2, be especially less than or equal to 2.5m/s
2, be especially less than or equal to 2m/s
2, be especially less than or equal to 1.5m/s
2, be especially less than or equal to 1m/s
2, be especially less than or equal to 0.5m/s
2gravity field in, carry out whole or in part the method.
In specific embodiment, be obviously different from the gravity field of intensity of gravity field at the earth's surface in intensity, carry out whole or in part the method according to this invention, the intensity of the gravity field of this earth surface equals 9.81m/s
2.
The meaning of " intensity is obviously different from the gravity field of the intensity of gravity field at the earth's surface " is intensity and 9.81m/s
2differ at least 5% gravity field.
Particularly advantageously b) utilize sound wave in step, this is for example, because in this case, the method can not be subject to gravity condition,, is subject under below-G conditions or carries out the remarkably influenced of the factor of the method under earth gravity condition.In fact, acoustic radiation force is produced by the interaction between sound field and material, and this point is irrelevant with gravity field.Therefore, for example, in stationary field, it is to be similar to have artificial gravity that material is guided to equilibrium theory of tide (for example,, towards node).
When in the time that step b) is utilized sound wave, unique difference is that the equilibrium theory of tide of the aggregate under 1g is a shade below the equilibrium theory of tide of the aggregate under 0g.
For example, under 0g, the equilibrium theory of tide of cell aggregation can be arranged on pressure node place, and under 1g, the equilibrium theory of tide of cell aggregation can be arranged on a shade below the node of the wall away from passage.
When being used in the intensity (g=9.81m/s of intensity lower than gravity field at the earth's surface
2) gravity field in time, the method according to this invention is particularly advantageous.In fact, for example, in the time utilizing sound wave (, ultrasonic standing wave), the sound power producing can make object (for example, wandering cell) assemble, and without any need for other external force.
Therefore, for example,, even when carry out the method according to this invention in the gravity field of low strength (, under weightlessness) time, the method also advantageously allows to obtain the compact 3D structure of object (for example, cell).
In specific embodiment, the present invention relates to method as defined above, wherein:
-liquid (L) is cell culture medium, and
At least one in the-the first aggregate (110) and the second aggregate (111) and optional the 3rd aggregate comprises cell, and
-in the time that described cell is present at least one in described the first aggregate (110), described the second aggregate (111) and optional described the 3rd aggregate, cultivate described cell.
Therefore, when carrying out when the method according to this invention, the cell being present at least one in the first aggregate and the second aggregate and optional the 3rd aggregate can be grown, and during cell cultures, the number of described cell is at least multiplied by 2 or be even multiplied by 5.
In a preferred embodiment, by cell cultures, form at least one tissue, especially, by cell cultures, form bone tissue.The tissue forming is preferably 3D tissue.
Preferably, be obviously different from intensity, especially in the gravity field lower than the intensity of gravity field at the earth's surface, carry out cell cultures.
Cell cultures under microgravity demonstrates double advantage.
First, for the tissue of fully growth provides in the buoyancy of ecotopia, as under normal physiological condition, the 3D that can grow under microgravity tissue.
Secondly, as mentioned above, be advantageously cosmonaut's the technology that needs development organizations engineering science, that is, bone is rebuild and tissue injury is replaced.
The present invention advantageously allows to assemble wandering cell under microgravity, and without any need for other external force, for example, to form compact 3D structure.
In a preferred embodiment, the first aggregate and the second aggregate and optional the 3rd aggregate are maintained at least one hour, especially at least one day, during especially at least one week in.
Particularly preferably be, apply sound wave to these first aggregates and the second aggregate and optional the 3rd aggregate, to form sound trap, wherein, stable aggregate can be kept to consolidate within a few hours, a couple of days or one number time.
In specific embodiment, during cell cultures, some cell culture mediums are introduced in passage at least one times.
In specific embodiment, during cell cultures, cell culture medium is updated at least once.The meaning of statement " cell culture medium is updated " is that the cell culture medium being again introduced in passage that the cell culture medium contacting with aggregate at first can have identical or different chemical property by another completely substitutes.
Because these aggregates are stable especially, therefore the present invention advantageously allows to upgrade cell culture medium, and do not disturb aggregate.This advantage attracts people's attention, and this is because when not utilizing time of the present invention, substratum can not be changed easily, in fact, under microgravity condition, flows and can produce the disturbance of taking aggregate out of.
Under microgravity, aggregate can be difficult for improving the shearing stimulation of tissue growth, this is because any disturbance produces the uncontrolled movement of structure.But in the present invention, can apply the vibration or the vibration that produce shear-stress, keep being configured in around 3D equilibrium theory of tide simultaneously.
In specific embodiment, the first aggregate and the second aggregate and optional the 3rd aggregate are 3D aggregate.
In specific embodiment, organize the first aggregate and the second aggregate and optional the 3rd aggregate more and exist along the longitudinal axis of passage.
In a preferred embodiment, many groups the one 3D aggregate and the 2nd 3D aggregate exist along the longitudinal axis of passage, and described many group aggregates are by performing step b) and obtain in the gravity field lower than the intensity of gravity field at the earth's surface in intensity.
the suspension of sound field producer, sound wave and object
Step b) and/or the sound wave that c) uses of step can have 10MHz or less, be preferably included in the primary frequency f between 0.5MHz and 10MHz
max.
In this range of frequency, use sound field producer can advantageously promote to maintain the integrity of viable cell or object (for example, vesica, liposome, bacterium or virus).
Step b) and/or the sound wave that c) uses of step can be advantageously along the thickness of passage and generate.
Sound field producer can be piezoelectrics, for example, and pottery.
For example, can use the Ferroperm Piezoceramics company by the Kvistgard of Denmark, the sound field producer of selling to number PZ26.
The part contacting with wall passage sound field producer can be circular or rectangle.
The area of this part can be greater than or less than aisle spare (, length is taken advantage of wide).According to forming the required configuration of aggregate, described area can equal aisle spare.
Sound field producer can provide power by positive string tension (tension).In modification, sound field producer can provide power by choppy sea tension force or square wave tension force.
Sound field producer can digitally or in analog be controlled.
Sound field producer can for example, provide power by wave producer (model 5062 of, being sold by the Tabor electronics company of Israel).
The ripple of launching by wave producer can be amplified by amplifier, and this amplifier is for example the model 9250 of being sold by the Tabor electronics company of Israel.
Step b) and/or step c) during, wave producer can produce the ripple of amplitude between 0Vp-p (volt peak-to-peak value) and 40Vp-p.
Step b) and/or step c) during produce acoustic density can be at 1J/m
3(joule/m
3) and 1000J/m
3between, for example, at 1J/m
3and 300J/m
3between, for example, at 5J/m
3and 50J/m
3between, can be for example 10J/m
3.
Step b) and/or the sound wave that c) uses of step can have for passage is along the primary frequency of the resonant frequency of a dimension in its transverse dimensions.
The transverse dimensions of passage is thickness and the width of passage.
The meaning of " for passage is along the primary frequency of the resonant frequency of a dimension in its transverse dimensions " is frequency f
0, make the transverse dimensions of the passage measured at the given position of the longitudinal axis along passage
zmeet
in formula, n is integer, and
c in formula
ffor example, for the velocity of sound in the liquid existing in passage, at the temperature (, 20 DEG C) of described liquid.
In other words, frequency f
0corresponding to theoretic frequency, in the given position of the longitudinal axis along passage, this theoretic frequency meets the resonant condition of sound wave in passage and the formation along the standing wave of considered transverse dimensions.
Step b) and/or the sound wave that c) uses of step can have at 0.5f
0and 1.5f
0between, especially at 0.75f
0and 0.95f
0between or at 1.05f
0and 1.25f
0between primary frequency.
Use this frequency can advantageously allow to produce enough strong to obtain the concentrated sound power of gratifying object.
Sound field producer can be fixed to one of them wall of passage.Suitably utilize any means known for skilled worker, especially, by bonding, can carry out this and fix.
The adaptive material of one deck acoustics can be present between at least one wall in sound field producer and conduit wall.
By using for the known any suitable material of skilled worker, can be made into acoustics adaptation.
In order to be created on step b) and/or step described sound wave c), can there are multiple sound field producers along the length of passage, sound field producer is preferably present on the same side of passage.
passage
According to the present invention, can use the passage described in US 2008/0067128, the content of US2008/0067128 is incorporated herein by reference.
Geometric properties
The width of passage and/or thickness can change at least a portion of its length, reduce alternatively.
Therefore,, in the time moving along the longitudinal axis of passage, the thickness of described passage can be constant or can change.Especially, passage can comprise at least two regions of axially following each other and presenting different thickness.
Passage, at least a portion of its length, especially, in its whole length, can present the thickness that is less than or equal to 3cm, better, is less than or equal to the thickness of 1cm.For example, passage is microchannel.
The meaning of " microchannel " is the passage in its whole length with the thickness that is less than or equal to 1cm.
Passage, at least a portion of its length, especially in its whole length, can be presented between 50 μ m and 3mm, the preferred thickness between 100 μ m and 500 μ m.
In the time moving along the longitudinal axis of described passage, the width of described passage can be constant or can change.For example, passage presents two regions of axially following each other and presenting different in width.
" longitudinal axis of passage " meaning is the line of the center of gravity of the cross section of interconnection passage.The longitudinal axis of passage can be straight or curve, and can be planar involved, and this plane can be some or the even whole symmetrical plane in the cross section of passage.
In embodiment, in the time observing from top, passage width can change, and passage can have cone shape.In this specific situation, sound field producer can be rectangle or be not rectangle.
In modification, in the time observing from top, passage can have the shape of the circle being connected by subchannel, especially linear subchannel.
Have in the configuration of shape of circle at passage, sound field producer can be columniform, and especially, described configuration can generate the aggregate of mosaic.
Passage at least a portion of its length, especially in its whole length, can have between 1mm and 40mm, the preferred width between 5mm and 20mm.
For example, along the length of the measured passage of its longitudinal axis between 3mm and 1000mm, preferably between 10mm and 500mm.
For example, passage has length, the width of 10mm and the thickness of 0.4mm of 100mm.
According to another illustrative embodiments, the length of passage can be between 10mm and 1000mm, and the width of passage can be between 1mm and 40mm, and the thickness of passage can be between 0.5mm and 3mm.
In the time moving along its longitudinal axis, passage can comprise constant substantially transverse section.
Passage, at least a portion of its length, especially, in its whole length, can have the transverse section of rectangle.
In modification, passage, at least a portion of its length, especially, in its whole length, can have foursquare or circular transverse section.
Passage can advantageously have the width/thickness ratio and/or the length/thickness ratio that are more than or equal to 10.
This ratio can advantageously prevent the three-dismensional effect in flow profile (profile).
In a preferred embodiment, passage, at least a portion of its length, especially, in its whole length, has the transverse section of rectangle and is more than or equal to 10 width/thickness ratio.
The wall of passage can be lamelliform.
The wall of passage, at least a portion of their length, especially, in their whole length, can have the thickness between 0.5mm and 5mm.
Passage, at least a portion of its length, can comprise the wall of variation in thickness.
This configuration can allow to produce sound wave joint or sound wave abdomen near the part of wall with different thickness, thereby aggregate is contacted with each other near this region.
When carrying out when the method according to this invention, can free oscillations at the wall on opposite of the wall that produces sound wave.
Entrance and exit
Passage can be with at least one, especially at least two inlet fluids are communicated with.
Passage can with at least one, especially at least two outlet fluids be communicated with.
Comprise multiple entrances and/or multiple outlet, for example, the passage of two entrances and two outlets, can be placed in stable framework, or is placed in the framework that can be tilted, to change universal gravity constant.
Feeder connection and/or outlet can be connected to syringe pump and/or peristaltic pump.In the time that feeder connection and/or outlet are connected to peristaltic pump, hydrodynamic damping device can be added between peristaltic pump and feeder connection and/or outlet.
Passage can be communicated with one or more outlet fluids, and in this exit, the multilayer aggregate forming by the method according to this invention can be discharged from.
Therefore, can open passage, to collect the multilayer aggregate forming.
Preferably, at least one entrance presents the width that is not less than passage width, and/or presents the cross section that is substantially rectangle.
In embodiments of the present invention, at least one entrance leads in a wall in diapire and the roof of passage, and another entrance leads in another wall in diapire and the roof of passage.
For example, two inlet faces are arranged towards each other.
In modification, all entrances lead to the diapire of passage or lead to the roof of passage.
In embodiments of the present invention, at least two entrances are relative to each other offset along the longitudinal axis of passage.
At least one entrance can be parallel to or lead to perpendicular to the longitudinal axis of passage passage substantially.
In embodiments of the present invention, at least one feed port is communicated with at least one inlet fluid by conduit, and this conduit especially comprises the pronged by-passing portions from conduit, the contiguous described tip of this feed port, especially leads to conduit perpendicular to conduit.
The by-passing portions of this conduit makes it start to form material sheet from feed points.
In embodiments of the present invention, at least the first outlet and the second outlet are communicated with passage fluid, and are separated from each other by the horizontal partition wall with the non-zero elevation measured along the thickness of passage.
Outlet can allow to reclaim the multilayer aggregate forming by the method according to this invention.
At least two outlets can relative to each other be offset along the longitudinal axis of passage.In modification, at least two outlets can be towards each other.
In embodiments of the present invention, passage is communicated with at least the first outlet, the second outlet and the 3rd outlet fluid, and the second outlet is disposed between the first outlet and the 3rd outlet along the thickness of passage.
In embodiments of the present invention, at least one outlet opening is communicated with the outlet fluid of passage by conduit, this conduit comprises that sectional side is to the part narrowing, especially the part of assembling towards tip, in the time observing from top, described part is trilateral, for example, this outlet opening is for example contiguous most advanced and sophisticated, especially leads to conduit perpendicular to conduit.
The convergence portion of this conduit can play the effect of avoiding forming at outlet opening place stagnation point.
Form the material of passage
The wall of passage can comprise the material being selected from below, is especially made up of the material being selected from below: synthetic glass or mineral glass, quartz, thermoplastic material (especially PMMA (polymethylmethacrylate) or polycarbonate) and metal.More generally, can use any material with acoustic impedance (, than at least 10 times, the acoustic resistance Chinese People's Anti-Japanese Military and Political College of fluid).
Passage can utilize the conventional manufacture method of the kind being used in microfluid field to manufacture.
In suitable situation, microchannel can be provided with at least one valve, for example, and magnetic valve.
liquid and object
Liquid can be biological liquid, as blood.
In modification, liquid can be water.
Liquid can be transparent for visible ray.
During step b), liquid can not flow.
According to embodiment, liquid at least step b) and/or step c) during flow, the Reynolds number of the stream of liquid is less than 10 alternatively.
Object can be single biomass cells or polydispersion biomass cells, especially hemocyte of disperseing, for example, and blood cell.
In specific embodiment, object has positive acoustics contrast factor.
At length definition " acoustics contrast factor " below.
Acoustic radiation force has expression formula below:
F
ac＝<E
ac>V
pkA(ρ，β)Sin(2ky)，
Wherein, <E
ac> is average acoustic energy density; V
pfor the volume of particulate matter, k=2 π/λ, wherein, λ is hyperacoustic wavelength
Acoustics contrast factor has expression formula below:
A(ρ，β)＝[(5ρ
p-2ρ
f)/(2ρ
p+ρ
f)]-β
p/β
f
Wherein:
-ρ
pand ρ
fbe respectively the density of particle p and the density of suspension f,
-β
pand β
fbe respectively the compression factor of particle p and the compression factor of suspension f, and
-y is the distance apart from standing wave nodal plane.
In specific embodiment, object has negative acoustics contrast factor.
In specific embodiment, object is selected from: bubble, microbubble, nano bubble, drop, adipocyte, blood fat, phospholipid capsule bubble or their mixture.
The object with negative acoustics contrast factor can b) be positioned in different pressure antinode places afterwards in step.In modification, the object with negative acoustics contrast factor can be positioned between pressure node and pressure antinode.
For example, the first object b) is located in the first pressure antinode place afterwards in step, the second object b) is located in the second pressure antinode place afterwards in step, described the second pressure antinode is different from the first pressure antinode and superposes with the first pressure antinode, described the second object is different from described the first object, for example, there is different density or different elastic constants.Described the first object and described the second object can have identical size.
In specific embodiment, the method is included in b) rear at least one feature of determining object of step, for example, and at least one acoustic feature, for example, the specific acoustic resistance of described object.
In specific embodiment, the method is included in the b) agglomeration step of rear object of step, in the time that described object is present in passage, especially, carries out whole or in part described cohesion.
In specific embodiment, step c) after, object is discharged to outside passage, for example, is discharged in container.
Object can be particle rigidity or deformable, for example, and granules of polystyrene.
More generally, object can be particle rigidity or deformable, polydispersion particle, biomass cells (especially hemocyte, for example, the cancer cells in blood sample or blood cell sample), bacterium, gluey emulsion or non-gluey emulsion, protein or liposome.
The mean sizes that is present in the object in passage can be less than or equal to 100 μ m.
The meaning of " mean sizes " is with the size of overall half statistics, is called as D50.
The sound power that the flow velocity adopting can be depending on the volume of handled sample, passage and applies.
For example, liquid can be with the flow velocity between 0.1ml/min to 100ml/min, during the method according to this invention all or part of, flows.
In specific embodiment, when the main sound power of maximum (acoustic primary force) and gravity have identical magnitude, (that is, being latex particle or the cell of 10 μ m for diameter, is 1pN (skin newton=10
-9)) time, the passage that is 1ml for volume, liquid can be with the flow rate of 0.1ml/min.
In described object is injected into passage time, the volume fraction of measured object can be 0.1% (v/v) or larger.The volume fraction of object is corresponding to [(volume of object)/(volume of the liquid that contains described object)] x100%.
Brief description of the drawings
According to consulting accompanying drawing and according to reading for implementing the below detailed description of limiting examples of the present invention, will understanding better the present invention, in accompanying drawing:
Fig. 1 illustrates the device for carrying out the method according to this invention,
Fig. 2 illustrates the aggregate that the method according to this invention obtains,
Fig. 3 is according to the view of the III-III of the passage using in Fig. 1,
The example of the multilayer aggregate that Fig. 4 forms according to the method shown in Fig. 1 for discharge,
Fig. 5 to Fig. 8 is the modification of the device for carrying out the method according to this invention,
Fig. 9 is the embodiment that can be used in the passage in the method according to this invention, and
Figure 10 to Figure 12 B illustrates the experimental result obtaining by the method according to this invention.
Embodiment
Fig. 1 illustrates the device 1 can be used in the method according to this invention.Device 1 comprises the passage 2 that axis X is extended along the longitudinal.
As mentioned above, passage 2 can be microchannel.
Passage 2 presents for example cross section for rectangle.In described example, the length/thickness ratio of passage 2 is greater than 10.
Passage 2 has diapire 3 and roof 4.As shown in the figure, passage 2 for example, is communicated with entrance 7 (, 5 entrances 7) fluid.
Entrance 7 is opened in the roof 4 of passage 2 and diapire 3, and as shown in the figure, 4 entrances 7 are opened perpendicular to X-axis line.In addition, the entrance 7 of opening in the roof 4 of passage 2 is parallel to X-axis line and leads to described passage 2.
In unshowned modification, entrance all leads to the diapire of passage.
Entrance 7 has the cross section of rectangle substantially, and has the width of the width that equals passage 2.
Entrance 7 is separated by the horizontal partition wall 10 perpendicular to longitudinal axis X.
As shown in the figure, entrance 7 presents the thickness of the thickness that is less than passage 2, for example, is less than the half of the thickness of passage 2.
In the embodiment shown in Fig. 1, some entrances 7 can be offset for example partition wall 10 along X-axis line.
As shown in the figure, at least one entrance 7 leads to the first area 45 that is connected to the second area 46 with larger thickness, and another entrance 7 leads to second area 46, along the thickness measurement thickness of passage 2.
Second area 46 is connected to the central zone 47 of passage 2.
Thickness difference between thickness difference between region 45 and region 46 and region 46 and region 47 is corresponding to the difference of altitude between partition wall 10.
As described below, device 1 can be used in the method according to this invention.
For example, as shown in Figure 1, carrier fluid L and multiple object O are passed entrance 7 and cause mobile.
Object O can be monodispersed or polydisperse, and described object O can be biomass cells, and liquid L can be biological liquid, for example, and blood.
In suitable situation, device 1 can have one or more valves that inject by entrance 7 for controlling object O, and wherein, this valve can be for example the magnetic valve that presents single passage or multiple passages.
In order to process large capacity object, can control object O by frequency and the flow velocity of the injection of entrance 7, to device 1 can be moved continuously.
This device 1, by water power focusing technology, provides object O (step a)) in the first overlap-add region 50 and second overlap-add region 60 of passage 2.
The first layer 51 of object O and the second layer 61 are presented on respectively in described the first overlap-add region 50 and the second overlap-add region 60, and the thickness of the thickness of described the first layer 51 and the second layer 61 focuses on to control by water power.
As shown in Figure 1, the first layer 51 of object O and the second layer 61 are separated by one deck neat liquid L.Being presented on object O in the first layer 51 can be with to be presented on object O in the second layer 61 identical.In modification, the object O being presented in the first layer 51 belongs to different kinds with the object O being presented in the second layer 61.In modification, the size that is presented on the object O in the first layer 51 is different from the size of the object O being presented in the second layer 61.
It can be disadvantageous water power shear-induced diffusion [3] that the layer of this object can stand for many suspensions undercurrent.
Therefore, device 1 is provided with the sound field producer 100a harmony field generator 100b of the roof 4 that is fixed to as shown in the figure passage 2.In Fig. 1, sound field producer 100a harmony field generator 100b arranges along passage 2, and is presented on the same side of described passage 2.In unshowned modification, sound field producer can be along access arrangement, and is presented on the opposite side of described passage.
In step b), sound field producer 100a can form the first aggregate 110 and the second aggregate 111 of object O.As shown in Figure 1, object O is placed in to suspend around the pressure node of the ripple being produced by sound field producer 100a more.In other words,, in the time that step b) finishes, each in the first aggregate 110 and the second aggregate 111 is positioned in different pressure node places.Sound field producer 100a can form and comprise staying wave frequency and moving of two node along the thickness of passage 2 with permission.In modification, sound field producer 100a does not produce standing wave along the thickness of passage 2, but can produce the sound field of force that can form aggregate.
In the time that acoustics manipulation attempts object to be placed in the equilibrium theory of tide of opposing gravity, adopt statement " acoustic levitation ".Position and the number of the node of acoustical behavior, sound power and the sound wave of object and suspension depended in equilibrium theory of tide.In the time of different equilibrium theory of tide (suspension) in passage of one species or different types of particle or aggregate, adopt statement " acoustics suspend (acoustic multi-levitation) " more.
Liquid L can flow during the method according to this invention, and can b) form the first aggregate 110 and the second aggregate 111 in step, and does not arrhea the period.
Acoustic focusing resists above-mentioned water power shear-induced diffusion.
Step b) can have a time length between 0.1s to 5 minute.
In unshowned other modification, the method according to this invention can form and comprise three layers or more multi-layered multilayer aggregate.
As mentioned above, the aggregate of object can be compacter than one deck object.Fig. 2 illustrates the top view of the first aggregate 110 obtaining in the time that step b) finishes according to the present invention.The first aggregate 110 comprises one group of object O contacting with each other, and for example, forms at least 50% can the contacting with each other of object O of described aggregate 110.
For example, according to the intensity of sound field and hydrodynamicparameter used, the present invention can form 2D aggregate and/or 3D aggregate.Provide hereinafter the definition of this 2D aggregate and 3D aggregate.
In addition,, in the time moving along Y-axis line, the first aggregate 110 comprises a string object O 110
1, it is corresponding to the displacement of the width along passage 2.In the time moving along the thickness of passage 2, the first aggregate 110 also comprises a string object O.Therefore, the first aggregate 110 is 3D aggregate.
In one embodiment, in the time moving along the width of passage, aggregate comprises a string object, but has the thickness being formed by least one object.In this case, aggregate is 2D aggregate.
Turn back to Fig. 1, now will describe process in accordance with the present invention example c) in detail.In Fig. 1, the first aggregate 110 contacts with the second aggregate 111, to form the multilayer aggregate 120 of object O.The acoustic wave action of inducing described the first aggregate 110 and the second aggregate 111 to move towards each other by the first aggregate 110 and the second aggregate 111 are subject to, and form described multilayer aggregate 120.
These sound waves are by forming the sound field producer 100b that comprises the staying wave frequency an of node and move and generate along the thickness of passage 2 with permission.In modification, sound field producer 100b does not produce standing wave along the thickness of passage 2, but can produce the sound field of force that can form multilayer aggregate.
In the example shown in Fig. 1, step c) comprises and makes aggregate 110 and aggregate 111 be subject to acoustic wave action, and the sound wave joint of this sound wave saves few than the sound wave of the sound wave of b) applying in step.In addition, shown in example in, the horizontal position of the sound wave that c) forms in step joint is different from the horizontal position that the sound wave of the sound wave of b) apply in step saves.
Therefore, aggregate 110 and aggregate 111 can be by the regions of the reduced number of node, cause formed aggregate again to be concentrated, thereby produce multilayer aggregate 120.
Step c) can have a time length between 10 seconds and 60 minutes.
As shown in Figure 3, can be present between sound field producer 100a and the roof 4 of passage 2 as the layer of gel 101 of specific acoustic resistance adapter.
Fig. 4 is illustrated in the discharge of the multilayer aggregate 120 forming in the illustrated method of Fig. 1.
In the embodiment shown in Fig. 4, passage 2 is communicated with multiple outlet 8 fluids.As shown in the figure, passage 2 can be connected to 3 outlets 8, these outlets in 8 two can towards each other and one can open, one can lead to the diapire 3 of passage 2, another can lead to roof 4, as shown in Figure 4.
The multilayer aggregate 120 forming can be discharged from by central outlet, and this point can expand to that tool haves three layers or more multi-layered multilayer aggregate.
Another embodiment of the method according to this invention has been shown in Fig. 5.In this accompanying drawing, for example, to form the first aggregate 110 and the second aggregate 111 with mode identical in Fig. 1 in the time that step b) finishes.
But, in this case, in the time not there is not sound wave, by making the first aggregate 110 and the second aggregate 111 be subject to action of gravity, described the first aggregate 110 is contacted with the second aggregate 111 and form multilayer aggregate 120.
In this example, the first aggregate 110 and the second aggregate 111 accessible areas 48, in this region 48,, especially, there is not sound field in the strength decreased of sound field in this region 48.Therefore, described aggregate 110 and aggregate 111 can be allowed to sedimentation, and form multilayer aggregate 120.
In modification, in step b), the first aggregate 110 and the second aggregate 111 can be after arrheaing the period and form, then, sound wave frequency and/or amplitude can be by tuning, to make aggregate 110 and aggregate 110 sedimentations to form thickness and the controlled multilayer aggregate 120 of composition.
In the example shown in Fig. 6, according to what describe in detail, form the first aggregate 110 and the second aggregate 111 in the embodiment shown in Fig. 1.But, the vicissitudinous thickness of diapire 3 tool of passage 2.In this example, the variation of the thickness of diapire 3 can make the sound wave being produced by sound field producer 100b have the node or the antinode that are positioned on diapire 3.In this case, during step c), the first aggregate 110 and the second aggregate 111 can be moved by the diapire 3 towards passage 2, and as shown in the figure, the second aggregate 111 can contact with described diapire 3.
The multilayer aggregate 120 forming can be positioned on the diapire 3 of passage 2.
Fig. 7 illustrates another embodiment, wherein, and the thickness of passage 2
tin at least a portion of its length, reduce, make in this way the sound wave joint of the sound wave of c) applying in step fewer than the sound wave joint of the sound wave of b) applying in step.Shown in example in, first sound field generator 100a and rising tone field generator 100b step b) and step c) send the sound wave with identical substantially primary frequency.
In unshowned modification, first sound field generator and rising tone field generator step b) and step c) send the sound wave with different primary frequencies.Difference between primary frequency can step c) form have different numbers, alternatively than the sound wave of the few sound wave of the sound wave of b) applying in step joint.
In unshowned modification, the thickness of passage increases at least a portion of its length, makes in this way the sound wave of c) applying in step compare the sound wave joint with different numbers from the sound wave of b) applying in step.
In unshowned modification, by increasing the number of node/antinode, can make aggregate contact and form the multilayer aggregate of described object.
Fig. 8 shows the possible configuration of the mobile multilayer aggregate 120 forming along passage 2.
A series of multilayer aggregates 120 can form along passage 2, then, flow and can be stopped.A series of wells 200 that are arranged on the wall 3 relative with rising tone field generator 100b with first sound field generator 100a can be used to hold multilayer aggregate 120.
Then the multilayer aggregate 120, forming utilizes the slow mobile well 200 that is transported to.The disposal system of this multilayer aggregate 120 can be manufactured in order.
Fig. 9 illustrates the embodiment of the passage 2 observing from top, and as shown in Figure 9, this passage 2 has the shape of circle 210, and this circle 210 is connected by passage 220.In the configuration of passage 220, sound field producer can be columniform, and can form the aggregate of mosaic.
example
Describe hereinafter the experimental result obtaining by the method according to this invention in detail.
Figure 10 shows the cancer cell 2D aggregate of the suspension obtaining in the passage of 250 μ m thickness.
Figure 11 A and Figure 11 B show many suspensions of particle.Diameter is the particle of the two kinds of types of 10 μ m: (two different positionss of 250 μ in m) are in equilibrium state at the thickness of passage for latex particle and silica dioxide granule.Figure 11 A illustrates these particles in the time that step b) finishes according to the present invention, and Figure 11 B illustrates these particles in the time that step c) finishes according to the present invention.
Figure 12 A illustrates and forms the suspend double-deck latex particle (dark color) of 10 μ m and two 2D layers of silica dioxide granule (light tone).
Figure 12 B illustrates the bilayer being made up of 3D latex particle aggregate and silica dioxide granule aggregate.
reference
[1] Hoyos etc., Fluidic separation device:US2008/0067128 A1,
[2] Callens etc., (2008) Analytical Chemistry 80,4866-4875,
[3] Williams etc., (2008) Analytical Chemistry 80,7105-7115,
[4] Kuznetsova etc., (2009) Biotechnology Progress 25 (3), 834-841.
Statement " comprising one/mono-" should be understood as that " comprising at least one ".
Statement " ... between " should be interpreted with comprised end points.
Claims (32)
1. a method that forms the multilayer aggregate (120) of object (O) in the passage that contains liquid (L) (2), described method comprises:
A) provide described object (O) in the first overlap-add region (50) and second overlap-add region (60) of described passage (2),
B), alternatively by apply transverse sound wave to described object (O) in each described region, preferably standing wave, obtains the first aggregate (110) and second aggregate (111) of described object (O), and
C) by making described the first aggregate (110) and described the second aggregate (111)
Zero in the time not there is not sound wave, is subject to action of gravity, or
Zero is subject to sound wave, the standing wave effect alternatively of inducing described the first aggregate (110) and described the second aggregate (111) to move towards each other,
Described the first aggregate (110) is contacted with described the second aggregate (111), and form the multilayer aggregate (120) of described object (O).
2. method according to claim 1, along the thickness of described passage (2) (
t) be created on step b) and/or described sound wave that step c) is used.
3. according to the method described in any one in claim 1 and 2, step c) comprises and makes described aggregate (110; 111) be subject to acoustic wave action, described sound wave has:
-different from the number of the sound wave joint of the sound wave of b) applying in step, save few sound wave than the sound wave of the sound wave of b) apply in step alternatively and save, and/or
-at least one sound wave joint, the horizontal position of described at least one sound wave joint is different from the horizontal position of the sound wave joint of the sound wave of b) applying in step, and/or
-at least one sound wave abdomen, the horizontal position of described at least one sound wave abdomen is different from the horizontal position of the sound wave abdomen of the sound wave of b) applying in step, and/or
-amplitude, described amplitude is less than the sound wave amplitude of b) applying in step.
4. according to method in any one of the preceding claims wherein, described passage (2) comprises the wall of variation in thickness and at least first sound field generator (100a) and the rising tone field generator (100b) that exist along the length of described passage (2) alternatively at least a portion of its length, and described first sound field generator (100a) and described rising tone field generator (100b) are respectively towards the wall part with different thickness of described passage.
5. according to the method described in last claim, there is at least first sound field generator (100a) and rising tone field generator (100b) along the length of described passage (2), and:
-described first sound field generator (100a) and described rising tone field generator (100b) step b) and step c) launch sound wave, and
-described passage (2) has the first wall and the second wall that lay respectively at described first sound field generator (100a) and described rising tone field generator (100b) opposite, described first wall and described the second wall have different thickness, make in this way the sound wave of c) applying in step have the node and/or the antinode that are positioned on described the second wall.
6. according to method in any one of the preceding claims wherein, the width of described passage (2) (
w) and/or thickness (
t) at least a portion of its length, change, reduce alternatively.
7. according to the method described in last claim, there is at least first sound field generator (100a) and rising tone field generator (100b) along the length of described passage (2), to be created on respectively step b) and step sound wave c), wherein:
-described first sound field generator (100a) and described rising tone field generator (100b) step b) and step c) launch the sound wave with identical substantially primary frequency, and
The width of-described passage (2) (
w) and/or thickness (
t) at least a portion of its length, change, reduce alternatively, the sound wave that makes in this way c) apply in step have number that the sound wave of the sound wave of b) apply from step saves different, save few sound wave than the sound wave of the sound wave of b) applying in step alternatively and save.
8. according to the method described in any one in claim 1 to 4 and 6, the primary frequency of the sound wave of c) applying in step is different from the primary frequency of the sound wave of b) applying in step, the sound wave that makes in this way c) to apply in step have the number of sound wave joint of the sound wave of b) applying from step different, save few sound wave than the sound wave of the sound wave of b) apply in step alternatively and save.
9. according to method in any one of the preceding claims wherein, comprising:
A) provide described object (O) in the first overlap-add region, the second overlap-add region and the 3rd overlap-add region of described passage (2),
B) alternatively by apply transverse sound wave, especially standing wave to described object (O) in each described region, obtain the first aggregate, the second aggregate and the 3rd aggregate of described object (O), and
C) by making described the first aggregate, described the second aggregate and described the 3rd aggregate
Zero in the time not there is not sound wave, is subject to action of gravity, or
Zero is subject to inducing the acoustic wave action of the power that described the first aggregate, described the second aggregate and described the 3rd aggregate move towards each other,
Described the first aggregate, described the second aggregate and described the 3rd aggregate are contacted, to form the multilayer aggregate of described object (O).
10. according to method in any one of the preceding claims wherein, wherein, in step a) in the first overlap-add region, the second overlap-add region and optional the 3rd overlap-add region (50 of described passage (2); 60) provide the layer (51 of described object (O); 61), described layer (51; 61) focus on and obtain by water power alternatively.
11. according to method in any one of the preceding claims wherein, wherein, a) being present in described object (O) in described first area in step has to be different from step and a) is present in size and/or the character of the described object (O) at least one region in described second area and/or described the 3rd region.
12. according to method in any one of the preceding claims wherein, and described object (O) is single biomass cells or polydispersion biomass cells, preferred mammal tumor cell line or Mammals non-tumor cell system, stem cell or primary cell line of disperseing.
13. according to method in any one of the preceding claims wherein, and described object (O) has positive acoustics contrast factor.
14. according to method in any one of the preceding claims wherein, and described object (O) has negative acoustics contrast factor.
15. according to the method described in last claim, and described object (O) is selected from: bubble, microbubble, nano bubble, drop, adipocyte, blood fat, phospholipid capsule bubble or their mixture.
16. according to method in any one of the preceding claims wherein, and described method is included in b) rear at least one feature of determining described object of step, for example, and at least one acoustic feature.
17. according to method in any one of the preceding claims wherein, described method is included in the b) agglomeration step of rear described object (O) of step, in the time that described object (O) is present in described passage (2), especially, carry out whole or in part described cohesion.
18. according to method in any one of the preceding claims wherein, step c) after, described object (O) is discharged to outside described passage, for example, is discharged in container.
19. according to method in any one of the preceding claims wherein, described the first aggregate (110) and described the second aggregate (111), and at least one in optional the 3rd aggregate comprises at least 100 objects (O), especially at least 250 objects (O), especially at least 500 objects (O), especially at least 750 objects (O), especially at least 1000 objects (O), especially at least 1500 objects (O).
20. according to method in any one of the preceding claims wherein, has multiple sound field producer (100a along the length of described passage (2); 100b), to be created on step b) and/or step described sound wave c), described sound field producer is preferably present on the same side of described passage (2).
21. according to method in any one of the preceding claims wherein, described liquid (L) at least step b) and/or step c) during flow, the Reynolds number of the stream of described liquid (L) is less than 10 alternatively.
22. according to method in any one of the preceding claims wherein, and described liquid (L) does not flow during step b).
23. according to method in any one of the preceding claims wherein, is less than or equal to 10m/s in intensity
2, be especially less than or equal to 9.5m/s
2, be especially less than or equal to 9m/s
2, be especially less than or equal to 8.5m/s
2, be especially less than or equal to 8m/s
2, be especially less than or equal to 7.5m/s
2, be especially less than or equal to 7m/s
2, be especially less than or equal to 6.5m/s
2, be especially less than or equal to 6m/s
2, be especially less than or equal to 5.5m/s
2, be especially less than or equal to 5m/s
2, be especially less than or equal to 4.5m/s
2, be especially less than or equal to 4m/s
2, be especially less than or equal to 3.5m/s
2, be especially less than or equal to 3m/s
2, be especially less than or equal to 2.5m/s
2, be especially less than or equal to 2m/s
2, be especially less than or equal to 1.5m/s
2, be especially less than or equal to 1m/s
2, be especially less than or equal to 0.5m/s
2gravity field in, carry out whole or in part described method.
24. according to method in any one of the preceding claims wherein, is obviously different from the gravity field of intensity of gravity field at the earth's surface in intensity, carries out whole or in part described method.
25. according to method in any one of the preceding claims wherein, wherein:
-described liquid (L) is cell culture medium, and
At least one in-described the first aggregate (110) and described the second aggregate (111) and optional the 3rd aggregate comprises cell, and
-in the time that described cell is present at least one in described the first aggregate (110), described the second aggregate (111) and optional described the 3rd aggregate, cultivate described cell.
26. according to the method described in last claim, by cell cultures, forms at least one tissue.
27. according to the method described in last claim, by cell cultures, forms bone tissue.
28. according to the method described in any one in claim 25 to 27, and during described cell cultures, some cell culture mediums are introduced in described passage (2) at least one times.
29. according to the method described in last claim, and during described cell cultures, described cell culture medium is updated at least once.
30. according to method in any one of the preceding claims wherein, and described the first aggregate and described the second aggregate and optional the 3rd aggregate are maintained at least one hour, especially at least one day, during especially at least one week in.
31. according to method in any one of the preceding claims wherein, and described the first aggregate (110) and described the second aggregate (111) and optional the 3rd aggregate are three-dimensional 3D aggregate.
32. according to method in any one of the preceding claims wherein, organizes described the first aggregate (110) and described the second aggregate (111) and optional the 3rd aggregate more and exists along the longitudinal axis of described passage (2).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP12154125.4 | 2012-02-06 | ||
| EP12154125.4A EP2623589A1 (en) | 2012-02-06 | 2012-02-06 | Method of forming a multilayer aggregate of objects |
| PCT/IB2013/050958 WO2013118053A1 (en) | 2012-02-06 | 2013-02-05 | Method of forming a multilayer aggregate of objects |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104114692A true CN104114692A (en) | 2014-10-22 |
| CN104114692B CN104114692B (en) | 2017-07-04 |
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ID=47997625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201380008319.2A Active CN104114692B (en) | 2012-02-06 | 2013-02-05 | The method for forming the multilayer aggregate of object |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US9719065B2 (en) |
| EP (2) | EP2623589A1 (en) |
| JP (1) | JP6173351B2 (en) |
| CN (1) | CN104114692B (en) |
| ES (1) | ES2691392T3 (en) |
| WO (1) | WO2013118053A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110255880A (en) * | 2019-05-16 | 2019-09-20 | 宿迁南航新材料与装备制造研究院有限公司 | A kind of preparation method of the super glass wool with super layer structure |
| CN111344557A (en) * | 2017-06-30 | 2020-06-26 | 法国国家科学研究中心 | Method and apparatus for manipulating objects |
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| JP6268669B2 (en) * | 2013-10-24 | 2018-01-31 | 株式会社Ihi | Apparatus and method for controlling sedimentation of foreign matter in liquid |
| US20150210979A1 (en) * | 2014-01-27 | 2015-07-30 | Northrop Grumman Systems Corporation | Scaffold-free tissue engineering using field induced forces |
| JP2015198619A (en) * | 2014-04-09 | 2015-11-12 | 大日本印刷株式会社 | Cell culture vessel, cell culture apparatus, and manufacturing method of cell structure |
| WO2016098011A1 (en) * | 2014-12-16 | 2016-06-23 | Celldynamics I.S.R.L. | Device for real time analysis of particles suspended in a fluid and method for the analysis of said particles |
| US10737012B2 (en) * | 2015-03-31 | 2020-08-11 | Biomet Biologics, Inc. | Cell washing using acoustic waves |
| AU2018320713A1 (en) | 2017-08-25 | 2020-02-06 | Ao Technology Ag | Surface acoustic wave (SAW) 3D printing method |
| EP3643406A1 (en) | 2018-10-24 | 2020-04-29 | Technische Universität München | Particle aggregation method and system in a channel |
| CN113908787A (en) * | 2021-11-19 | 2022-01-11 | 西安交通大学 | Micro-reaction channel structure and acoustic micro-reactor and chemical production system based on same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111344557A (en) * | 2017-06-30 | 2020-06-26 | 法国国家科学研究中心 | Method and apparatus for manipulating objects |
| CN111344557B (en) * | 2017-06-30 | 2023-04-07 | 法国国家科学研究中心 | Method and apparatus for manipulating objects |
| CN110255880A (en) * | 2019-05-16 | 2019-09-20 | 宿迁南航新材料与装备制造研究院有限公司 | A kind of preparation method of the super glass wool with super layer structure |
Also Published As
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| WO2013118053A1 (en) | 2013-08-15 |
| EP2812428B1 (en) | 2018-07-25 |
| ES2691392T3 (en) | 2018-11-27 |
| JP6173351B2 (en) | 2017-08-02 |
| US9719065B2 (en) | 2017-08-01 |
| CN104114692B (en) | 2017-07-04 |
| US20150037863A1 (en) | 2015-02-05 |
| EP2812428A1 (en) | 2014-12-17 |
| JP2015506181A (en) | 2015-03-02 |
| EP2623589A1 (en) | 2013-08-07 |
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